1. Field of Invention
The present disclosure relates to methods, compositions, and kits for purifying, detecting, and characterizing nucleic acids.
2. Description of Related Art
The identification of the presence or absence of specific nucleic acid sequences in a sample is a central part of many assays and tests used in the modern research lab and clinical setting. In the typical scheme, the nucleic acids from the sample are first separated from other macromolecules present in the sample by manipulating various physical properties. For example, nucleic acids typically bear a net negative charge at neutral pH, owing to the phosphodiester backbone. This property can be manipulated to separate nucleic acids from other macromolecules using anion exchange resins. As another example, differential solubility of nucleic acids compared to other macromolecules in certain solvents is used to extract nucleic acids from the sample. Numerous other such schemes exist. However, the amount of target nucleic acid relative to the total amount of nucleic acid purified typically is very low. Therefore, some type of amplification is necessary before the target nucleic acid can be detected. Either the amount of specific nucleotide sequence(s) is increased by target amplification methods such as polymerase chain reaction (PCR) or the specific nucleotide sequence(s) is/are reacted with a detectable label and the signal from the label is amplified to detectable levels.
Existing nucleic acid detection assays are usually based on PCR that detects the small portion of genome. The most advanced amplification format is real-time PCR that is performed in ‘closed tube format’ eliminating the risk of amplicon contamination. Unfortunately, these methods have limited utility. One limitation is that target-specific amplification methods such as PCR are inherently error-prone. For example, although the stringency of primer hybridization can be controlled, there nonetheless exists the potential for non-specific primer binding and primer-independent amplification, which can lead to false-positive results. Moreover, different sequences can amplify at different rates, resulting in amplification bias. As a result, quantitative analysis of multiple nucleic acid sequences in a single reaction often suffers from a lack of sensitivity, resulting in limited multiplex capability. The limitation of this approach is limited multiplex capability of the PCR and potential false negative results if the amplicon region is mutated. Another requirement is that real-time PCR requires complicated thermocycling equipment which could limit its applicability to automated systems. In addition, target nucleic acids that are present at low concentrations relative to other nucleic acids may be effectively “masked” from the polymerase, which could result in false-negative results. Other factors may exist that reduce both the specificity and sensitivity of such assays.
Therefore, methods and compositions are needed for specific and sensitive isolation and analysis of at least one target nucleic acid containing at least one specific sequence.